Summary/Abstract Transfusion-associated acute lung injury (TRALI), currently the leading cause of transfusion- associated fatality, is triggered by transfusion of blood products containing leukocyte antibodies and/or biological response modifiers to susceptible patients. Although many different leukocyte antibodies have been shown to induce TRALI, those specific for the leukocyte antigen HNA-3a are especially prone to cause very severe, and often fatal reactions. Unfortunately, it has not been possible to screen blood donors routinely for anti-HNA-3a because its molecular properties have for many years eluded investigators and because HNA-3a was thought to be neutrophil-specific, a cell difficult to use in serologic studies. Thus, very little is known about the prevalence of anti-HNA-3a in blood donors and the immunogenicity of this antigen is poorly understood. Even less is known about antibodies that recognize HNA-3b, the allele of HNA-3a, although they should, in theory, be as likely to cause TRALI as anti-HNA-3a. We recently showed that HNA-3a is carried on choline transporter- like protein 2 (CTL2) and that the HNA-3a/b polymorphism is almost certainly determined by an R>Q154 (R=HNA-3a, Q = HNA-3b) amino acid substitution in the first extracellular loop of the 10- membrane-spanning CTL2 protein. These findings suggest ways to develop a practical assay for detection of anti-HNA-3a and anti-3b suitable for routine donor screening. In this application, we propose to generate recombinant and synthetic CTL2 and CTL2 fragments containing R154 and Q154 and characterize their reactions against a panel of HNA-3-specific antibodies. Constructs found to be most sensitive and specific for anti-CTL2 detection will be produced in quantity, formatted into a prototype solid phase immunoassay, and used to screen 7,000 serum samples from transfused, non-transfused and parous males and females to determine the prevalence of anti-HNA-3a in these populations. The Q154 version of these constructs should provide comparable information about anti-HNA-3b. Findings made are expected to 1) define, for the first time, the prevalence of anti-HNA-3a and anti- HNA-3b in blood donor populations; 2) characterize the immunogenicity of HNA-3 and HNA-3b (likelihood of an antibody being induced upon exposure to antigen through transfusion or during pregnancy) and 3) establish a basis for determining whether routine screening of blood donors for anti-HNA-3a and anti-HNA-3b is warranted.